This project will develop a strategy to detect levels of hydroxymethylation (hmC) in genomic DNA. We will first combine a method to selectively attach a biotin to hmC with our demonstrated technique of detecting single biotin modifications in DNA to demonstrate the efficacy of our approach and optimize experimental conditions. We will then apply the technique to clinical tissue samples. First, we will measure the global hmC content of normal breast tissue and of breast carcinoma and compare our findings with results from conventional approaches. Finally, we will utilize the capability of our approach to isolate modified DNA selectively and apply the system to identifying alterations in the hmC content of a fragment of the LZTS1 gene, an important tumor suppressor.